Molecular characterization of the RNA-binding protein, TRN-1 in the lepidopteran insects

TRN-1是一种与哺乳动物T细胞胞内抗原-1同源的RNA结合蛋白，在鳞翅目昆虫、家蚕和果蝇的各个组织中都有表达。与TIA-1家族相似，TRN-1分子在N端具有三个重复的潜在RNA识别基序(RRMS)，在C端具有辅助结构域，通过研究外源重组蛋白和转录本的产生，发现TRN-1分子具有转录调控蛋白的功能。等，基因320，67-79,2003)。通过共聚焦荧光显微镜分析过表达GFP-TRN-1融合蛋白和缺乏C端辅助结构域和RRM结构域的GFP-TRN-1细胞，研究了家蚕TRN-1各结构域在培养细胞中的亚细胞定位和作用，以期更深入地了解TRN-1的细胞功能。重组GFP-RRMS融合蛋白具有一定的聚(U)RNA结合活性。GFP-TRN-1缺失C端辅助结构域和GFP-C端结构域可能定位于胞核，而全长GFP-TRN-1定位于胞核和胞浆。然而，AcNPV感染细胞后，GFP-TRN-1在细胞核内有明显的定位。此外，GFP-TRN-1的过表达可导致重组杆状病毒产生的荧光素酶活性在感染过程中发生变化，从而表明鳞翅目昆虫TRN-1可能控制杆状病毒和细胞转录产物对病毒感染引起的应激反应的功能。

TRN-1, an RNA-binding protein homologous with mammalian T-cell intracellular antigen-1 has been found to express in every tissue of the Iepidopteran, Bombyx mori and Spodoptera frugiperda. TRN-1 molecule has been shown to possess the domain structure consisting of three-repetitive potential RNA-recognition motifs (RRMs) in the N-terminal followed by auxiliary domain in the C-terminal as similar with TIA-1 family, and to function as a modulator protein for transcripts by investigating the production of foreign recombinant proteins and transcripts in the Bombyx TRN-1 knock-down BmN4 cells (Kotani, et. al., Gene 320, 67-79, 2003). Subcellular localization and role of each domain of the Bombyx TRN-1 in the cultured cells has been investigated by the confocal fluorescence microscopy analyses on the cells over-expressing GFP-TRN-1 fusion protein and GFP-TRN-1 lacking C-terminal auxiliary domain and RRM domain in order to gain more insight into cellular function of the TRN-1. The recombinant GFP-fusion proteins with RRMs were indicated to assuredly have the poly (U) RNA binding activity. It was also shown that GFP-TRN-1 lacking C-terminal auxiliary domain and GFP-C-terminal domain were likely to be localized in the nucleus, while full-length GFP-TRN-1 was localized both in the nucleus and cytoplasm. However, clear localization of GFP-TRN-1 in the nucleus was observed after AcNPV was infected into the cells. In addition, it was indicated that GFP-TRN-1 over-expression could cause change of the recombinant baculovirus-produced luciferase activity during its infection, thus demonstrating that lepidopteran TRN-1 may control the function of both baculovirus and cellular transcripts in the response to stress caused by the viral infection.